This invention relates to a method and apparatus for correlating particles and their characteristics, and more particularly to a method and apparatus for correlating a number of separately detected particle characteristics and the particles.
In the fields of particle analysis and particle study it is desirable to ascertain as many characteristics as possible for each particle to be studied. The more characteristics that are determined, the more reliably a machine can distinguish one particle from another somewhat similar particle. This is especially important in areas such as blood analysis where it is desired to distinguish various types of blood cells. The visual classification of blood cells is usually based upon such parameters as color, size and shape of the nucleus after appropriate staining, and the granularity, color and relative quantity of the stained cytoplasm.
Automatic recognition and classification of cells is being pursued today. One method, known as pattern recognition, involves laying out the unknown cells upon a surface or substrate for automatic examination under a high resolution microscope. The microscope viewing field is scanned by some transducer, for example, a vidicon, that responds to the display. The field is scanned and information bits are stored representing the optically detected features of the cells. A computer suitably programmed in accordance with algorithms devised to enable cell identification processes the data from the viewing field in an attempt to match the information with similar information in its memory. This method requires the processing of thousands of bits of information and involves expensive equipment including computers with large storage banks. Notwithstanding the expense and complexity of the equipment involved, absolute accuracy in recognition and classification is not guaranteed, and generally recognition time is quite slow.
A second method, known as a flow-through or simply flow system method, is exemplified by the apparatus in U.S. Pat. No. 2,656,508 which shows a Coulter Counter.RTM.. The mark Coulter Counter is a registered trademark, No. 995,825, of Coulter Electronics, Inc. of Hialeah, Fla. In this method, very rapid measurements are made upon each cell in a liquid suspension as the suspension is flowed through an electrically excited sensing zone. Sensing zones may also be excited by light of one or more colors. The gross parameters which can be measured by this general method are cell volume or size, DNA content, RNA content, color, fluorescence, absorption of light, etc. Sensing zones which measure these gross parameters make what are termed low resolution measurements. Electrically excited sensing zones may utilize not only DC but high frequency AC, as described in U.S. Pat. No. 3,502,974. U.S. Pat. No. 3,497,690 shows a flow through system which combines several measurements in the same sensing zone. Another example of a flow system is given in U.S. Pat. No. 3,960,449 which yields a measurement having to do with size, shape, and internal structure of each cell. These systems are capable of performing a somewhat greater number of measurements and are referred to as intermediate resolution measurements. Flow systems have the great advantage of high speed, measuring in excess of one thousand cells per second, but can only make a limited number of low resolution measurements on each cell.
If it were possible to obtain an increased number of measurements of diverse properties of cells than is presently possible, the probability of correctly identifying specific cells would be greatly enhanced. As each sensing zone is capable of only a low number of measurements several must be used. It is necessary to perform each and every measurement on each cell in order to achieve the ultimate goal of total correlation of all measurements with the cell on which measurements were made. To do this, it is necessary to arrange several sensing zones in series, i.e., in tandem so that the same cell passes through each sensing zone. U.S. Pat. No. 3,822,095 by Hirschfeld is an example of such multiple sensing zones. The problem that arises that the measurements made by the successive sensing zones are not simultaneous. Hence, in order to collate the measurements for classifying the cells, the results of the first-made measurements have to be stored until the last is made.
It is difficult to build other types of sensing zones such that they are less than several centimeters distant from each other. The flow rates used in flow-through systems are commonly on the order of five to ten meters per second and hence it takes from one to two milliseconds for each particle to progress one centimeter down the flow chamber. If mechanical considerations dictate that the sensing zones be spread out over a distance of five centimeters, then the total delay between the first and last sensing zones will be of the order of five to ten milliseconds. To have the particle suspension sufficiently dilute, such that the first sensing zone were precluded from measuring a particle until the last sensing zone had completed its measurement, would require that the minimum spacing between particles would be five centimeters, which corresponds to one-half to one millisecond. The average spacing, considering that the particles are randomly distributed in the suspension, would have to be greatly in excess of five centimeters. Such a weak concentration of particles and very long intervals between particles would vitiate the advantages of the flow system. On the other hand, if the high speed sensing capabilities of flow systems are to be retained, it must be possible to have many particles in such a five centimeter long flow chamber.
Increasing the particle concentration of the sample suspension to utilize fully the high-speed capability of the flow-through method means that the upstream sensing zone will have observed many subsequent particles in the time it takes for a given particle to progress from the first sensing zone to the last. This being the case, the results of measurements of the upstream sensing zones must be stored for later comparison with the measurements at the downstream zone. A problem now presented is how can the machine reliably ascribe the measurements made at the various sensing zones to the proper particle? The obvious solution would be to match the measurement made by the first sensing zone on the very first particle of an aliquot of sample with the very first measurement made at the subsequent and at the last sensing zones, the second with the second, etc., ad infinitum. This would entail storing the first measurement for perhaps a millisecond, but the match would still be made. If the stream of particle-bearing sample were then laid down on an examining surface for subsequent microscopic examination, measurements could be ascribed to the correct particles either by ensuring that the first particle to flow through the flow chamber is the first particle on the examining surface or by the technique disclosed in U.S. Pat. No. 3,924,947.
As is explained in U.S. Pat. No. 3,924,947, depending on each sensing zone to recognize a "first" particle would be risky, and in a device for recognizing types of white cells, or malignant cells, upon which a life-or-death diagnostic decision may be made, no compromise can be tolerated. The risk stems from two sources. First, the sensing zones respond to different properties of the cells. If a first sensing zone responded to fluorescence and a second sensing zone to volume, and the first particle had no fluorescence, the fluorescence of the second particle would be ascribed to the first particle to go through the second sensing zone. The erroneous correlation would then make all following matches erroneous.
Secondly, the practical difficulties of beginning a sample run at the exact instant a first particle entered the first sensing zone, despite hydraulic and electronic starting transients, if not insuperable, would require enormously delicate, fast, and accurate apparatus.
It would seem that the most direct method would be to measure the flow rate of the sample through the flow chamber, and, knowing the distance between the sensing zones, to delay the correlation of measurements by the ratio of that distance to the flow rate. However, the "flow rate" is only the average flow rate. The actual flow in the flow chamber must be laminar to maintain the particle stream. Minor inaccuracies in the centering of the particle stream due to imperfections or dirt on the inner walls of the flow chamber cause minor variations in the delay. Also, the velocity on center is somewhat faster than the average velocity. A fixed delay cannot be relied upon, either.
The flow-through or on-stream method enables some physical separation into groups of cells, exemplified by the apparatus of Fulwyler, U.S. Pat. No. 3,380,584. Thus, cells in each group may be examined independently. Until recently it had not been deemed possible to correlate measurements made on specific cells during an on stream analysis with cells which were measured by pattern recognition methods. U.S. Pat. No. 3,924,947, issued to the inventor of the present application describes an apparatus for correlating the cell characteristics measured on-stream with the cells which are to be measured or have been measured using pattern recognition techniques. The noted patent describes the correlation of information obtained at one sensing zone with the particle locations for the corresponding particles on a substrate.
To summarize, if a number of characteristics are to be ascertained by flow-through technique, different types of sensing zones are employed, each of which detects at least a particular characteristic of the particle. Generally these sensing zones are positioned in series along a flow stream in a manner such as is taught in U.S. Pat. No. 3,822,095.
In such an apparatus, the various sensing zones located along the fluid stream are closely spaced with respect to one another. As each sensing zone is closely spaced to a preceding or succeeding sensing zone, such that their measurements are almost simultaneous, the amount of jitter or randomness in particle arrival time between two adjacent sensing zones is small so that it is simple to correlate particle arrival times and particle characteristics measured at adjacent sensing zones. However, if the various sensing zones are not extremely closely spaced, more sophisticated tactics are called for to ensure that all measurements of a particular particle are in fact ascribed to that particular particle.
If, after passing through the flow chamber bearing several sensing zones, the particles are to be laid out on a substrate for subsequent microscopic examination, a flow chamber-to-substrate correlation must be made. There is no problem in correlating the characteristics measured by the sensing zone closest to the substrate to which the particle is passed with the characteristic ascertained by means of the position of the particle on the substrate. This correlation is taught in the incorporated patent. However, if the sensing zone is not the closest sensing zone to the substrate it is difficult to correlate the characteristic ascertained by that sensing zone with the particle location on the substrate. The difficulty encountered is due to the fact that the flow stream itself is not absolutely stable. That is, there is some jitter or erratic random movement in the flow stream so that the precise timing of the particle movement from each zone to a substrate positioned some distance away is difficult if not impossible to predict. Consequently, correlation with the particle location on the substrate may prove extremely difficult and beyond the capabilities of machines and programs currently available.
The present invention teaches a method for permitting many particles between the first and last sensing zones of such a tandem arrangement of sensing zones without losing track of the identity of each particle and permitting all of the measurements on each particle to be correctly ascribed to that particle. The techniques used, while seeking a different end, are related to the principles upon which the aforementioned U.S. Pat. No. 3,924,947, is based.